Antibodies to post-translationally modified insulin in type 1 diabetes

Abstract

Aim/hypothesis Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. Methods oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAAwas measured using the gold-standard radiobinding assay (RBA). Results MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; a nd g l y c at i o n of Lys 2 9 a n d P h e 1 i n ch a i n B . Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin.

Antibodies to post-translationally modified insulin as a novel biomarker for prediction of type 1 diabetes in children

“…Insulin was chemically modified as previously described to generate oxPTM-INS modified by HOCl, • OH and glycation using ribose [11]. Hen egg lysozyme (HEL; Sigma-Aldrich, Milan, Italy) was similarly modified and used as a control antigen.…”

“…Being different from the native proteins, the post-translational modification (PTM) of self-antigens may be recognised as foreign and result in breakdown of tolerance [8–10]. Oxidative stress is a key feature of many autoimmune diseases and results in an excess of reactive oxidants able to generate oxidative PTM (oxPTM) [4, 11]. Products of oxidative stress are increased in type 1 diabetes [12] and also in individuals at risk [13].…”

“…During insulitis, the beta cells that are under stress and the high influx of metabolically active immune cells generate large quantities of reactive oxidants, including the superoxide radical, hydroxyl radical ( • OH), hypochlorous acid (HOCl) and peroxynitrate [14]. We have shown that autoimmunity to insulin may result from neoepitopes induced by oxPTM [11]. We found that antibodies specific to oxidative post-translationally modified insulin (oxPTM-INS) are present in the majority of newly diagnosed individuals with type 1 diabetes and are significantly more abundant than autoantibodies to native insulin (NT-INS) [11].…”

“…We have shown that autoimmunity to insulin may result from neoepitopes induced by oxPTM [11]. We found that antibodies specific to oxidative post-translationally modified insulin (oxPTM-INS) are present in the majority of newly diagnosed individuals with type 1 diabetes and are significantly more abundant than autoantibodies to native insulin (NT-INS) [11]. …”

“…To address this we have developed an ELISA that detects auto-reactivity to oxPTM-INS. We showed that the assay detecting antibodies to oxPTM-INS (oxPTM-INS-Ab) is highly accurate (84% sensitivity, 99% specificity), may detect over 30% of individuals who are negative to the RBA for IAA and, when combined with IAA, identifies 95% of individuals with newly diagnosed type 1 diabetes [11]. However, the predictive potential of oxPTM-INS-Ab is not known yet.…”